Canine leishmania vaccine

ABSTRACT

The present invention provides vectors that contain and express in vivo  Leishmania  KMP11 or an epitope thereof that elicits an immune response in a dog against  Leishmania , compositions comprising said vectors, methods of vaccination against  Leishmania , and kits for use with such methods and compositions.

INCORPORATION BY REFERENCE

This application claims benefit of U.S. provisional patent applicationSer. No. 60/866,848 filed Nov. 21, 2006.

All documents cited or referenced herein (“herein cited documents”), andall documents cited or referenced in herein cited documents, togetherwith any manufacturer's instructions, descriptions, productspecifications, and product sheets for any products mentioned herein orin any document incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention.

FIELD OF THE INVENTION

The present invention relates to the field of vaccine againstLeishmaniasis, specifically against canine Leishmaniasis.

BACKGROUND ART

Leishmaniasis is a major and severe parasitic disease of humans, canids(dogs, wolves, foxes, coyotes, jackals), and felids (lions, tigers,domestic cats, wild cats, other big cats, and other felines includingcheetahs and lynx).

The agent of leishmaniasis is a protozoan parasite and belongs to theleishmania donovani complex. This parasite is widely distributed intemperate and subtropical countries of Southern Europe, Africa, Asia,South America and Central America (Desjeux P., Trans. R. Soc. Trop. Med.Hyg., 2001, 95: 239-43). Leishmania donovani infantum (L. infantum) isresponsible for the feline and canine disease in Southern Europe,Africa, and Asia. In South America and Central America, the agent isLeishmania donovani chagasi (L. chagasi), which is closely related to L.infantum. In humans, the agent is Leishmania donovani donovani (L.donovani), which is closely related to L. infantum and L. chagasi.

The parasite is transmitted to humans, felids and canids by sand flies,which species vary depending on the geographic location. Phlebotomusariasi (P. ariasi) and Phlebotomus perniciosus (P. perniciosus) are thecarriers most common in Southern Europe, Africa, and Asia, whereasLutzomyia longipalpis (L. longipalpis) is most common in Southern andCentral America.

The domestic reservoir of Leishmaniasis are dogs, which may suffer froma severe disease characterized by chronic evolution of viscero-cutaneoussigns occurring in less than 50% of infected animals (Lanotte G. et al.,Ann. Parasitol. Hum. Comp., 1979, 54: 277-95). On the other hand, bothasymptomatic and symptomatic dogs with detectable antibodies can beinfectious to phlebotomine vectors (Molina R. et al., Trans. R. Soc.Trop. Med. Hyg., 1994, 88: 491-3; Courtenay O. et al., J. Infect. Dis.,2002, 186: 1314-20). Cats can be carriers of the protozoan parasites andare considered as secondary potential reservoirs.

These parasites cause visceral leishmaniasis and/or cutaneousleishmaniasis. Visceral leishmaniasis results in clinical symptoms likefever, cachexia, hepatosplenomegaly (enlargement of the liver andspleen), and blood cytopenia. Cutaneous leishmaniasis occurs in varyingpresentations, from the self-limited and even self-healing cutaneousforms to fatal systemic disease. Lesions of cutaneous leishmaniasis mayoccur anywhere on the body but the most common sites are those which areexposed to the environment and are therefore more susceptible to bitesfrom the sand flies. The initial papule rapidly gives rise to an ulcer.Systemic leishmaniasis is rare but is invariably fatal if not treatedpromptly. Systemic leishmaniasis affects the internal body organs,specifically the spleen and the liver.

In canines, the disease is associated with cutaneous symptoms or withvisceral symptoms or both cutaneous and visceral symptoms, and is lethalin the absence of therapy.

Numerous treatments have been described but none is fully satisfactorydue to toxicity of the treatment itself or a tendancy for the animal torelapse.

Mass detection of seropositive dogs followed by culling and/or drugtreatment, or the mass application of deltamethrin-impregnated collars,was shown to have an impact in reducing human and canine Leishmaniasisprevalence in endemic areas of Southern Europe, Africa, and Asia (MaroliM. et al., Med. Vet. Entomol., 2001, 15: 358-63; Mazloumi Gavgani A. S.et al., Lancet, 2002, 360: 374-9), although the efficacy of eliminatingseropositive canines has been debated (Dietze R. et al., Clin. Infect.Dis., 1997, 25: 1240-2; Moreira Jr. E. D. et al., Vet. Parasitol., 2004,122: 245-52). These control measures are either considuer unacceptable,expensive or not effective (Gradoni L. et al., Vaccine, 2005, 23:5245-51).

Mathematical models used to compare the effectiveness of various toolsfor controlling Leishmaniasis suggest that a canine vaccine may be themost pratical and effective method (Dye C., Am. J. Trop. Med. Hyg.,1996, 55: 125-30). Therefore, the development of vaccines able toprotect canids from leishmaniasis and/or to prevent disease progressionin infected animals, is highly desirable for the implementation ofLeishmaniasis control programs as well for the veterinary community(Gradoni L. et al., Vaccine, 2005, 23: 5245-51).

The state of the art is best summarized in US patent applicationUS-A-2006/0194753. This document describes a vaccine containing a DNAexpression vector encoding L. infantum KMP11 (kinetoplastid membraneprotein 11) protein. However, the experimental results on mice (as shownin FIG. 1 of 2006/0194753) showed that mice vaccinated with pMCV1.4plasmids expressing KMP11 had worse results than the control mice as tothe presence of lesions within 8 weeks after challenge infection (lesionscores of about 3.2 and about 1.6 for vaccinated and control mice,respectively). Furthermore, experiments on dogs in FIG. 2 of2006/0194753 show that after administration of a mixture of onerecombinant pMOK plasmid expressing L. infantum p36 antigen and threerecombinant pMCV1.4 plasmids expressing L. infantum TSA (thiol-specificantioxidant protein), L. infantum gp63 and L. infantum KMP11 antigens,no antibodies were detectable. There was no clear difference between theresults of the vaccinated group and those of the control group. Afterchallenge infection with 10^(7.7) L. infantum promastigotes, the numberof infected dogs in the vaccinated group showed only a slight differenceto the control group.

Basu et al. (Basu R. et al., J. Immunol., 2005, 174: 7160-71) describedan experiment using golden hamsters immunized with KMP11 containingpCMV-LIC mammalian expression vector versus control animals immunizedwith a blank vector construct not harboring KMP11 (pCMV-LIC). Animals ofboth groups received two intramuscular administrations to the hind legthigh muscle (using a 28-guage needle), given 8 days apart, of 100 μg ofplasmids dissolved in saline. On day 15, a lethal parasite challenge wasdone with either of two strains, L donovani AG83 or L. donovani GE1F8R.All of the vaccinated hamsters immunized with KMP11 DNA survived thelethal challenge of AG83 and GE1F8R and remained healthy until thetermination of the experiment at 8 months postinfection, whereas allnon-immunized and blank vector-immunized hamsters succumbed to virulentL. donovani challenge within 6 months.

Currently, no vaccine is available for Leishmania-susceptible subjects,including for canids.

SUMMARY OF THE INVENTION

Accordingly, the present invention relates to an innovative vaccinestrategy, which is based on Leishmania KMP11 (kinetoplastid membraneprotein 11) antigen in order to prevent diffusion and implantation ofthe parasite into internal organs.

It is therefore an object of this invention to provide a vaccine capableof protecting subjects (i.e., canids, felids, and humans) fromleishmaniasis and/or to prevent disease progression in infectedsubjects.

It is also an object of the present invention to provide methods ofusing such vaccines in order to protect canids from leishmaniasis and/orto prevent disease progression in infected canids.

It is also an object of the present invention to provide methods ofusing such vaccines in order to protect felids from leishmaniasis and/orto prevent disease progression in infected felids.

It is also an object of the present invention to provide methods ofusing such vaccines in order to protect humans from leishmaniasis and/orto prevent disease progression in infected humans.

It is therefore additionally an object of this invention to provide invivo expression vectors encoding Leishmania KMP11 antigen or immunogenor an epitope thereof for such vaccines and/or such methods of use.

It is noted that in this disclosure and particularly in the claims,terms such as “comprises”, “comprised”, “comprising” and the like canhave the meaning attributed to it in U.S. Patent law; e.g., they canmean “includes”, “included”, “including”, and the like; and that termssuch as “consisting essentially of” and “consists essentially of” havethe meaning ascribed to them in U.S. Patent law, e.g., they allow forelements not explicitly recited, but exclude elements that are found inthe prior art or that affect a basic or novel characteristic of theinvention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF DRAWINGS

The following detailed description, given by way of example, and whichis not intended to limit the invention to specific embodimentsdescribed, may be understood in conjunction with the accompanyingfigures, incorporated herein by reference, in which:

FIG. 1 shows the frequency of CD8 before vaccination, between an initialvaccination and a subsequent boost vaccination, and afterboost-vaccination; expressed in percentage of CD8 cells present in theperipheral blood.

FIG. 2 shows the frequency of IFN-gamma-producing cells beforevaccination, between an initial vaccination and a subsequent boostvaccination, and after boost-vaccination; expressed in percentage ofIFN-gamma+lymphocytes present in the peripheral blood.

FIG. 3 shows the specific CD8 T cells proliferation before vaccination,between an initial vaccination and a subsequent boost vaccination, andafter boost-vaccination; expressed in percentage of specific anti-KMP11CD8 T cells in the CD8 T cells population.

FIG. 4 shows the specific CD4 T cells proliferation before vaccination,between an initial vaccination and a subsequent boost vaccination, andafter boost-vaccination; expressed in percentage of specific anti-KMP11CD4 T cells in the CD4 T cells population.

FIG. 5 is a Western Blot showing the IgG response of a KMP11 vaccinateddog, where column 1 is unvaccinated, column 2 is KMP11 vaccinated dogpost-priming (D14), column 3 is KMP11 vaccinated dog post-boost (D41),column 4 is KMP11 vaccinated dog post-boost (D55), column 5 is KMP11vaccinated dog post-boost (D76), column 6 is control dog post-priming(D14) and column 7 is control dog post-boost (D41). The study utilizedL. infantum antigens 6 μg/104, first antibodies (serum) 1/1000 andsecond antibodies (anti-dogs IgG-HRP) 1/2000.

FIG. 6 is the plasmid diagram of the ALVAC donor plasmid pJSY1992.1.

FIG. 7 shows a theoretical restriction enzyme gel for the genomic DNA ofvCP2350.1.1.5, created in Vector NTI.

FIG. 8 is a restriction analysis gel of vCP2350.1.1.5 after digestionwith BgIII, HindIII and PstI, and separation by 0.8% agarose gelelectrophoresis.

FIG. 9 is a Southern blot analysis of vCP2350.1.1.5 obtained by probingwith Leishmania synthetic KMP11 probe.

FIG. 10 is the plasmid diagram of the donor plasmid VR2001-TOPA orVR2001-TOPO.

The sequences SEQ ID No 1 and SEQ ID No 2 show the nucleic acid sequenceof L. infantum KMP11 of the strain of used in the examples and the aminoacid sequence of the protein encoded by this nucleic acid sequence,respectively.

The sequence SEQ ID No 3 shows the codon-optimized nucleic acid sequenceencoding the KMP11 protein of L. infantum as presented in NCBI GenBANKdatabase accession number CAA64883 and in SEQ ID No 4.

The sequence SEQ ID No 5 shows the nucleic acid sequence of one strandof the plasmid pVR1020KMP11.

The sequence SEQ ID No 6 shows the nucleic acid sequence of one strandof the ALVAC donor plasmid pJSY1992.1.

DETAILED DESCRIPTION

In one aspect, the present invention relates to a vaccine strategy,which is based on a prime-boost administration regimen, where theprimo-administration and the boost administration(s) utilize acomposition comprising a pharmaceutically or veterinary acceptableexcipient, diluent or vehicle and an in vivo expression vectorcomprising a polynucleotide sequence, that contains and expresses theLeishmania antigen KMP11 or immunogen or epitopes thereof, as describedherein.

Leishmania KMP11 antigens are derived from, for example, L. infantum orL. chagasi. KMP11 is a highly conserved surface membrane protein presentin all members of the family Kinetoplastidae, and is differentiallyexpressed both in amastigote and promastigote forms of Leishmania(Jardim A. et al., Biochem. J., 1995, 305: 315-20; Jardim A. et al.,Biochem. J., 1995, 305: 307-13; Berberich C. et al., Biochim. Biophys.Acta, 1998, 1442: 230-7). The nucleic acid sequence of the gene and theamino acid sequence of the protein KMP11 of Leishmania are available inpublicly accessible databases, notably as L. infantum in the GenBankdatabase under the accession numbers X95627 and X95626. The nucleic acidseqeucne of L. donovani is also available from the GenBank database,notably under the accession number S77039.

The present invention relates to the use of in vivo expression vectorsin a prime-boost administration regimen, comprising aprimo-administration of a vaccine comprising, in a pharmaceuticallyacceptable vehicle, diluent or excipient, an in vivo expression vectorcontaining a polynucleotide sequence for expressing, in vivo, LeishmaniaKMP11 polypeptide, antigen, epitope or immunogen, followed by a boostadministration of a vaccine comprising, in a pharmaceutically acceptablevehicle or excipient, an in vivo expression vector containing apolynucleotide sequence for expressing, in vivo, Leishmania KMP11antigen, epitope or immunogen to protect canids, felids and humans fromleishmaniasis and/or to prevent disease progression in infected canids,felids and humans.

By definition, a prime-boost regimen comprises at least oneprimo-administration and at least one boost administration using atleast one common polypeptide, antigen, epitope or immunogen. The vaccineused in primo-administration can different in nature from those used asa later booster vaccine. The primo-administration may comprise one ormore administrations. Similarly, the boost administration may compriseone or more administrations.

In a further aspect, the present invention relates to a vaccinecomposition comprising a pharmaceutically or veterinarily acceptableexcipient, diluent or vehicle and an in vivo expression vectorcomprising a polynucleotide sequence, which contain and express theLeishmania antigen KMP11 or immunogen or epitopes thereof, as describedbelow.

As used herein, the term “antigen” or “immunogen” means a substance thatinduces a specific immune response in a host animal. The antigen maycomprise a whole organism, killed, attenuated or live; a subunit orportion of an organism; a recombinant vector containing an insert withimmunogenic properties; a piece or fragment of DNA capable of inducingan immune response upon presentation to a host animal; a protein, apolypeptide, a peptide, an epitope, a hapten, or any combinationthereof. Alternately, the immunogen or antigen may comprise a toxin orantitoxin.

The term “immunogenic protein or peptide” as used herein also refersincludes peptides and polypeptides that are immunologically active inthe sense that once administered to the host, it is able to evoke animmune response of the humoral and/or cellular type directed against theprotein. Preferably the protein fragment is such that it hassubstantially the same immunological activity as the total protein.Thus, a protein fragment according to the invention comprises orconsists essentially of or consists of at least one epitope or antigenicdeterminant. The term epitope relates to a protein site able to inducean immune reaction of the humoral type (B cells) and/or cellular type (Tcells).

The term “immunogenic protein or peptide” further contemplatesdeletions, additions and substitutions to the sequence, so long as thepolypeptide functions to produce an immunological response as definedherein. In this regard, particularly preferred substitutions willgenerally be conservative in nature, i.e., those substitutions that takeplace within a family of amino acids. For example, amino acids aregenerally divided into four families: (1) acidic—aspartate andglutamate; (2) basic—lysine, arginine, histidine; (3) non-polar—alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine,tryptophan; and (4) uncharged polar—glycine, asparagine, glutamine,cystine, serine threonine, tyrosine. Phenylalanine, tryptophan, andtyrosine are sometimes classified as aromatic amino acids. It isreasonably predictable that an isolated replacement of leucine withisoleucine or valine, or vice versa; an aspartate with a glutamate orvice versa; a threonine with a serine or vice versa; or a similarconservative replacement of an amino acid with a structurally relatedamino acid, will not have a major effect on the biological activity.Proteins having substantially the same amino acid sequence as thereference molecule but possessing minor amino acid substitutions that donot substantially affect the immunogenicity of the protein are,therefore, within the definition of the reference polypeptide.

The term “epitope” refers to the site on an antigen or hapten to whichspecific B cells and/or T cells respond. The term is also usedinterchangeably with “antigenic determinant” or “antigenic determinantsite”. Antibodies that recognize the same epitope can be identified in asimple immunoassay showing the ability of one antibody to block thebinding of another antibody to a target antigen.

An “immunological response” to a composition or vaccine is thedevelopment in the host of a cellular and/or antibody-mediated immuneresponse to a composition or vaccine of interest. Usually, an“immunological response” includes but is not limited to one or more ofthe following effects: the production of antibodies, B cells, helper Tcells, and/or cytotoxic T cells, directed specifically to an antigen orantigens included in the composition or vaccine of interest. Preferably,the host will display either a therapeutic or protective immunologicalresponse such that resistance to new infection will be enhanced and/orthe clinical severity of the disease reduced. Such protection will bedemonstrated by either a reduction or lack of symptoms normallydisplayed by an infected host, a quicker recovery time and/or a loweredviral titer in the infected host.

The terms “immunogenic” protein or polypeptide as used herein alsorefers to an amino acid sequence which elicits an immunological responseas described above. An “immunogenic” protein or polypeptide, as usedherein, includes the full-length sequence of the protein, analogsthereof, or immunogenic fragments thereof. By “immunogenic fragment” ismeant a fragment of a protein which includes one or more epitopes andthus elicits the immunological response described above. Such fragmentscan be identified using any number of epitope mapping techniques, wellknown in the art. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996). For example,linear epitopes may be determined by e.g., concurrently synthesizinglarge numbers of peptides on solid supports, the peptides correspondingto portions of the protein molecule, and reacting the peptides withantibodies while the peptides are still attached to the supports. Suchtechniques are known in the art and described in, e.g., U.S. Pat. No.4,708,871; Geysen et al., 1984; Geysen et al., 1986, all incorporatedherein by reference in their entireties. Similarly, conformationalepitopes are readily identified by determining spatial conformation ofamino acids such as by, e.g., x-ray crystallography and 2-dimensionalnuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra.Methods especially applicable to the proteins of T. parva are fullydescribed in the PCT Application Serial No. PCT/US2004/022605incorporated herein by reference in its entirety.

Synthetic antigens are also included within the definition, for example,polyepitopes, flanking epitopes, and other recombinant or syntheticallyderived antigens. See, e.g., Bergmann et al., 1993; Bergmann et al.,1996; Suhrbier, 1997; Gardner et al., 1998. Immunogenic fragments, forpurposes of the present invention, will usually include at least about 3amino acids, preferably at least about 5 amino acids, more preferably atleast about 10-15 amino acids, and most preferably about 15-25 aminoacids or more amino acids, of the molecule. There is no critical upperlimit to the length of the fragment, which could comprise nearly thefull-length of the protein sequence, or even a fusion protein comprisingat least one epitope of the protein.

Accordingly, a minimum structure of a polynucleotide expressing anepitope is that it comprises or consists essentially of or consists ofnucleotides to encode an epitope or antigenic determinant of aninfluenza protein or polyprotein. A polynucleotide encoding a fragmentof the total protein or polyprotein, more advantageously, comprises orconsists essentially of or consists of a minimum of 15 nucleotides, atleast 15-30, advantageously about 30-45 nucleotides, and preferablyabout 45-75, at least 57, 87 or 150 consecutive or contiguousnucleotides of the sequence encoding the total protein or polyprotein.Epitope determination procedures, such as, generating overlappingpeptide libraries (Hemmer et al., 1998), Pepscan (Geysen et al., 1984;Geysen et al., 1985; Van der Zee R. et al., 1989; Geysen, 1990;Multipin® Peptide Synthesis Kits de Chiron) and algorithms (De Groot etal., 1999), and in PCT Application Serial No. PCT/US2004/022605 all ofwhich are incorporated herein by reference in their entireties, can beused in the practice of the invention, without undue experimentation.Other documents cited and incorporated herein may also be consulted formethods for determining epitopes of an immunogen or antigen and thusnucleic acid molecules that encode such epitopes.

The term “vaccine composition” or “vaccine” covers herein anycomposition able, once it has been injected to a subject, includingcanids, felids and humans, to protect the subject from cutaneousleishmaniasis and/or visceral leishmaniasis, including to preventimplantation of the parasite, and/or to prevent disease progression ininfected subjects, and/or to limit the diffusion of runaway parasites tointernal organs. This may be accomplished by the vaccine through theinduction of humoral immune response against KMP11, notably theinduction of anti-KMP11 IgG1, and/or through the induction ofcell-mediated immune response against KMP11, including through theinduction of a CD8 cell-mediated immune response against KMP11 and/orthe induction of a CD4 cell-mediated immune response against KMP11. Theinduction of a humoral immune response is desirable in instances ofcutaneous leishmaniasis, and the induction of cell-mediated immune isdesirable in instances of response in case of visceral leishmaniasis. Anadvantage of the vaccine strategy and of the use of the vaccines of thepresent invention is to induce both humoral immune response andcell-mediated immune response, which permits protection of the subjectfrom both cutaneous leishmaniasis and visceral leishmaniasis.

The pharmaceutically or veterinary acceptable excipient, diluent orvehicle may be water, saline or a buffer, or another substance known tothose of skillin the art and recognized by those of skill in the art asan acceptable excipient.

In a further aspect, the present invention relates to an in vivoexpression vector comprising a polynucleotide sequence, which containsand expresses the Leishmania antigen KMP11 or immunogen or epitopesthereof, as described herein.

The in vivo expression vector includes any transcription unit containinga polynucleotide or a gene of interest and those essential elements forits in vivo expression. These expression vectors can be plasmids orrecombinant viral vectors.

As used herein, the term “polynucleotide” includes DNA and RNA, andderivatives thereof, such as those containing modified backbones. Itshould be appreciated that the invention provides polynucleotidescomprising sequences complementary to those described herein.

Polynucleotides according to the invention can be prepared in differentways (e.g. by chemical synthesis, by gene cloning etc.) and can takevarious forms (e.g. single stranded, double stranded, primers, probesetc.) (see Maniatis et al., Molecular Cloning: a Laboratory Manuel, ColdSpring Harbor Laboratory, 1982).

The polynucleotide is generally an open reading frame (ORF), startingfrom a start codon (methionine codon) and ending with a terminationsignal (stop codon). The polynucleotide can also include regions thatregulate its expression, such as transcription initiation, translationand transcription termination. Thus, also included are promoters andribosome binding regions (in general these regulatory elements lieapproximately between 60 and 250 nucleotides upstream of the start codonof the coding sequence or gene; Doree S M et al., J. Bacteriol. 2001,183(6): 1983-9; Pandher K et al., Infect. 1 mm. 1998, 66(12): 5613-9;Chung J Y et al., FEMS Microbiol letters 1998, 166: 289-296),transcription terminators (in general the terminator is located withinapproximately 50 nucleotides downstream of the stop codon of the codingsequence or gene; Ward C K et al., Infect. 1 mm. 1998, 66(7): 3326-36).In the case of an operon, such regulatory regions may be located agreater distance upstream of the gene or coding sequence.

As used herein, the term “derivative” refers to a polypeptide, or anucleic acid encoding a polypeptide, that has one or more conservativeamino acid variations or other minor modifications such that (1) thecorresponding polypeptide has substantially equivalent function whencompared to the wild type polypeptide or (2) an antibody raised againstthe polypeptide is immunoreactive with the wild-type polypeptide.

The term “conservative variation” denotes the replacement of an aminoacid residue by another biologically similar residue, or the replacementof a nucleotide in a nucleic acid sequence such that the encoded aminoacid residue does not change or is another biologically similar residue.Examples of conservative variations include the substitution of onehydrophobic residue such as isoleucine, valine, leucine or methioninefor another hydrophobic residue, or the substitution of one polarresidue for another polar residue, such as the substitution of argininefor lysine, glutamic for aspartic acid, or glutamine for asparagine, andthe like. The term “conservative variation” also includes the use of asubstituted amino acid in place of an unsubstituted parent amino acidprovided that antibodies raised to the substituted polypeptide alsoimmunoreact with the unsubstituted polypeptide.

More generally, the present invention encompasses polynucleotidederivative. As used herein, the term “derivative” refers to apolypeptide, or a nucleic acid encoding a polypeptide, that has at leastabout 50% identity, at least about 60% identity, at least about 70%identity, at least about 75% identity, at least about 80% identity, atleast about 85% identity, 90% identity, at least about 95% identity, andat least about 96%, 97%, 98%, or 99% or more identity to the amino acidsequence SEQ ID No 2. Sequences having such homology or identity canencompass genetic code degeneration. The percentage of identity betweentwo amino acid sequences can be established by the NCBI (National Centerfor Biotechnology Information) pairwise blast and the blosum62 matrix,using the standard parameters (see, e.g., the BLAST or BLASTX algorithmavailable on the “National Center for Biotechnology Information” (NCBI,Bethesda, Md., USA) server, as well as in Altschul et al. J. Mol. Biol.1990. 215. 403-410; and thus, this document speaks of using thealgorithm or the BLAST or BLASTX and BLOSUM62 matrix by the term“blasts”).

“Code” as used herein does not mean that the polynucleotide is limitedto an actual coding sequence but also encompasses the whole geneincluding its regulatory sequences which are non-coding sequences.

Sequence homology or identity such as nucleotide sequence homology alsocan be determined using the “Align” program of Myers and Miller,(“Optimal Alignments in Linear Space”, CABIOS 4, 11-17, 1988,incorporated herein by reference) and available at NCBI, as well as thesame or other programs available via the Internet at sites thereon suchas the NCBI site.

Alternatively or additionally, the term “homology” or “identity”, forinstance, with respect to a nucleotide or amino acid sequence, canindicate a quantitative measure of homology between two sequences. Thepercent sequence homology can be calculated as:(N_(ref)−N_(dif))*100/N_(ref), wherein N_(dif) is the total number ofnon-identical residues in the two sequences when aligned and whereinN_(ref) is the number of residues in one of the sequences. Hence, theDNA sequence AGTCAGTC will have a sequence identity of 75% with thesequence AATCAATC (N_(ref)=8; N_(dif)=2).

Alternatively or additionally, “homology” or “identity” with respect tosequences can refer to the number of positions with identicalnucleotides or amino acids divided by the number of nucleotides or aminoacids in the shorter of the two sequences wherein alignment of the twosequences can be determined in accordance with the Wilbur and Lipmanalgorithm (Wilbur and Lipman, 1983 PNAS USA 80:726, incorporated hereinby reference), for instance, using a window size of 20 nucleotides, aword length of 4 nucleotides, and a gap penalty of 4, andcomputer-assisted analysis and interpretation of the sequence dataincluding alignment can be conveniently performed using commerciallyavailable programs (e.g., Intelligenetics™ Suite, Intelligenetics Inc.CA). When RNA sequences are said to be similar, or have a degree ofsequence identity or homology with DNA sequences, thymidine (T) in theDNA sequence is considered equal to uracil (U) in the RNA sequence.Thus, RNA sequences are within the scope of the invention and can bederived from DNA sequences, by thymidine (T) in the DNA sequence beingconsidered equal to uracil (U) in RNA sequences.

Advantageously, sequence identity or homology such as amino acidsequence identity or homology can be determined using the BlastP program(Altschul et al., Nucl. Acids Res. 25, 3389-3402, incorporated herein byreference) and available at NCBI, as well as the same or other programsavailable via the Internet at sites thereon such as the NCBI site.

The following documents (each incorporated herein by reference) providealgorithms for comparing the relative identity or homology of sequencessuch as amino acid residues of two proteins, and additionally oralternatively with respect to the foregoing, the teachings in thesereferences can be used for determining percent homology or identity:Needleman S B and Wunsch C D, “A general method applicable to the searchfor similarities in the amino acid sequences of two proteins,” J. Mol.Biol. 48:444-453 (1970); Smith T F and Waterman M S, “Comparison ofBio-sequences,” Advances in Applied Mathematics 2:482-489 (1981); SmithT F, Waterman M S and Sadler J R, “Statistical characterization ofnucleic acid sequence functional domains,” Nucleic Acids Res.,11:2205-2220 (1983); Feng D F and Dolittle R F, “Progressive sequencealignment as a prerequisite to correct phylogenetic trees,” J. of Molec.Evol., 25:351-360 (1987); Higgins D G and Sharp P M, “Fast and sensitivemultiple sequence alignment on a microcomputer,” CABIOS, 5: 151-153(1989); Thompson J D, Higgins D G and Gibson T J, “ClusterW: improvingthe sensitivity of progressive multiple sequence alignment throughsequence weighing, positions-specific gap penalties and weight matrixchoice,” Nucleic Acid Res., 22:4673-480 (1994); and, Devereux J,Haeberlie P and Smithies 0, “A comprehensive set of sequence analysisprogram for the VAX,” Nucl. Acids Res., 12: 387-395 (1984). And, withoutundue experimentation, the skilled artisan can consult with many otherprograms or references for determining percent homology.

The vaccines according to the instant invention include vectors encodingat least the KMP11 polynucleotide or gene from L. infantum and/or theKMP11 polynucleotide or gene from L. chagasi, and/or the KMP11polynucleotide or gene from L. donovani. Advantageously, for SouthernEurope, Africa and Asia, vaccines include vectors encoding the KMP11polynucleotide or gene from at least L. infantum. Advantageously, forSouthern and Central Americas, vaccines include vectors encoding theKMP11 polynucleotide or gene from at least L. chagasi.

Codon preference among different species can be dramatically different.To enhance the expression level of a foreign protein, it is important tomatch the codon frequency of the foreign protein to that of the hostexpression system (Kim et al., Gene, 1997, 199(1-2): 293-301). For codonoptimization, other factors than codon frequency can be taken intoconsideration, e.g. DNA motifs and repeats, secondary structure, GCcontent, repetitive codons, restriction endonuclease sites, functionalmotifs like splice site or terminator structure. Algorithms have beencreated to facilitate the design of the optimal nucleotide sequence.Geneart GmbH (Regensburg, Germany) has developed the proprietaryGeneOptimizer™ software (WO-A-04/059556 and WO-A-06/013103) thatimplements multi-parameter optimization in one single operation. Takinginto account the most important parameters in parallel, the softwaregenerates a total of up to 500,000 optimized variants of the targetsequence in an evolutionary approach and selects the one that is bestsuited. It has been reported that such optimized genes have up to a100-fold increase in expression yields compared to the original genesequence (Bradel-Tretheway et al., J. Virol. Methods, 2003, 111(2):145-56; Disbrow et al., Virology, 2003, 311(1): 105-14).

The published nucleic acid sequences for KMP11 protein of L. infantum(NCBI GenBank database accession number CAA64883) were optimized by theGeneOptimizer™ software.

The codon-optimized synthetic nucleic acid sequence for KMP11 protein ofL. infantum is designated as SEQ ID No 3. The codon-optimized nucleicacid sequences encode a polypeptide having the same amino acid sequenceas those disclosed in GenBank CAA64883, also designated SEQ ID No 4. Thecodon-optimization changes only the nucleic acid sequence and not theencoded amino acid sequence.

A further object of the present invention relates to a codon-optimizedpolynucleotide sequence encoding a Leishmania KMP11 antigen. Oneembodiment of this invention is the codon-optimized polynucleotidesequence SEQ ID No 3, encoding a L. infantum KMP11 antigen SEQ ID No 4.

Another object relates to an in vivo expression vector comprising acodon-optimized polynucleotide sequence encoding a Leishmania KMP11antigen. An embodiment of this object is an in vivo expression vectorcomprising the codon-optimized polynucleotide sequence SEQ ID No 3,encoding a L. infantum KMP11 antigen SEQ ID No 4.

More generally, the present invention encompasses in vivo expressionvectors including any plasmid (EP-A2-1001025; Chaudhuri P Res. Vet. Sci.2001, 70(3), 255-6) containing the polynucleotide or gene of LeishmaniaKMP11 and elements necessary for its in vivo expression.

As used herein, the term “plasmid” includes any DNA transcription unitcomprising a polynucleotide according to the invention and the elementsnecessary for its in vivo expression in a cell or cells of the desiredhost or target; and, in this regard, it is noted that a supercoiled ornon-supercoiled, circular plasmid, as well as a linear form, areintended to be within the scope of the invention.

In a specific, non-limiting example, the pVR1020 or pVR1012 plasmid(VICAL Inc.; Luke C. et al., Journal of Infectious Diseases, 1997, 175,91-97; Hartikka J. et al., Human Gene Therapy, 1996, 7, 1205-1217) orpAB110 (U.S. Pat. No. 6,852,705) can be utilized as a vector for theinsertion of a polynucleotide sequence. The pVR1020 plasmid is derivedfrom pVR1012 and contains the human tPA signal sequence. Each plasmidcomprises or contains or consists essentially of, the polynucleotideaccording to the present invention, operably linked to a promoter orunder the control of a promoter or dependent upon a promoter, whereinthe promoter is advantageously adjacent thereto the polynucleotide ofthe present invention. In general, it is advantageous to employ a strongpromoter that is functional in eukaryotic cells. One example of a usefulpromoter is the immediate early cytomegalovirus promoter (CMV-IE) ofhuman or murine origin, or optionally having another origin such as therat or guinea pig. The CMV-IE promoter can comprise the actual promoterpart, which may or may not be associated with the enhancer part.Reference can be made to EP-A-260 148, EP-A-323 597, U.S. Pat. Nos.5,168,062, 5,385,839, and 4,968,615, as well as to PCT Application NoWO-A-87/03905. The CMV-IE promoter is advantageously a human CMV-IE(Boshart M. et al., Cell, 1985, 41, 521-530) or murine CMV-IE. In moregeneral terms, the promoter has either a viral or a cellular origin. Astrong viral promoter other than CMV-IE that may be usefully employed inthe practice of the invention is the early/late promoter of the SV40virus or the LTR promoter of the Rous sarcoma virus. A strong cellularpromoter that may be usefully employed in the practice of the inventionis the promoter of a gene of the cytoskeleton, such as e.g. the desminpromoter (Kwissa M. et al., Vaccine, 2000, 18, 2337-2344), or the actinpromoter (Miyazaki J. et al., Gene, 1989, 79, 269-277). Functional subfragments of these promoters, i.e., portions of these promoters thatmaintain adequate promoter activity, are included within the presentinvention, e.g. truncated CMV-IE promoters according to PCT ApplicationNo. WO-A-98/00166 or U.S. Pat. No. 6,156,567 can be used in the practiceof the invention. A promoter useful in the practice of the inventionconsequently includes derivatives and sub fragments of a full-lengthpromoter that maintain an adequate promoter activity and hence functionas a promoter, adventageously having promoter activity substantiallysimilar to that of the actual or full-length promoter from which thederivative or sub fragment is derived, e.g., akin to the activity of thetruncated CMV-IE promoters of U.S. Pat. No. 6,156,567 in comparison tothe activity of full-length CMV-IE promoters. Thus, a CMV-IE promoter inthe practice of the invention can comprise or consist essentially of orconsist of the promoter portion of the full-length promoter and/or theenhancer portion of the full-length promoter, as well as derivatives andsub fragments.

Advantageously, the plasmids comprise or consist essentially of otherexpression control elements. It is especially advantageous toincorporate stabilizing sequence(s), e.g., intron sequence(s), forexample, the first intron of the hCMV-IE (PCT Application No.WO-A-89/01036), the intron II of the rabbit β-globin gene (van Ooyen etal., Science, 1979, 206, 337-344). As to the polyadenylation signal(polyA) for the plasmids and viral vectors other than poxviruses, usecan more be made of the poly(A) signal of the bovine growth hormone(bGH) gene (see U.S. Pat. No. 5,122,458), or the poly(A) signal of therabbit β-globin gene or the poly(A) signal of the SV40 virus.

In one embodiment of the present invention, the plasmid vector ispVR1020KMP11, as described in example 1 herein.

More generally, the present invention encompasses in vivo expressionvectors including any recombinant viral vector containing thepolynucleotide or gene of Leishmania KMP11 and all those elementsnecessary for its in vivo expression.

Said recombinant viral vectors could be selected from, for example, thepoxviruses, especially avipox viruses, such as fowlpox viruses orcanarypox viruses. In one embodiment, the fowlpox virus is a TROVAC (seeWO-A-96/40241). In another embodiment, the canarypox vector is an ALVAC.The use of these recombinant viral vectors and the insertion ofpolynucleotides or genes of interest is fully described in U.S. Pat. No.5,174,993; U.S. Pat. No. 5,505,941 and U.S. Pat. No. 5,766,599 forfowlpox, and in U.S. Pat. No. 5,756,103 for canarypox. More than oneinsertion site inside the viral genome could be used for the insertionof multiple genes of interest.

In one embodiment the viral vector is an adenovirus, such as a humanadenovirus (HAV) or a canine adenovirus (CAV).

In another embodiment the viral vector is a human adenovirus,specifically a serotype 5 adenovirus, rendered incompetent forreplication by a deletion in the E1 region of the viral genome,especially from about nucleotide 459 to about nucleotide 3510 byreference to the sequence of the hAd5 disclosed in Genbank under theaccession number M73260 and in the referenced publication Chroboczek etal, 1992. The deleted adenovirus is propagated in E1-expressing 293(Graham et al., 1977) or PER cells, especially PER.C6 (Falloux et al.,1998). The human adenovirus can additionally or alternatively be deletedin the E3 region, especially from about nucleotide 28592 to aboutnucleotide 30470. The deletion in the E1 region can be done incombination with a deletion in the E3 region (see, e.g. Shriver et al.,2002; Graham et al., 1991; Ilan et al., 1997; U.S. Pat. Nos. 6,133,028and 6,692,956; Tripathy et al., 1994; Tapnell, 1993; Danthinne et al.,2000; Berkner, 1988; Berkner et al., 1983; Chavier et al., 1996). Theinsertion sites can be the E1 and/or E3 loci (region) eventually after apartial or complete deletion of the E1 and/or E3 regions.Advantageously, when the expression vector is an adenovirus, thepolynucleotide to be expressed is inserted under the control of apromoter functional in eukaryotic cells, such as a strong promoter,adventageously a cytomegalovirus immediate-early gene promoter (CMV-IEpromoter), especially the enhancer/promoter region from about nucleotide−734 to about nucleotide +7 in Boshart et al., 1985 or theenhancer/promoter region from the pCI vector from Promega Corp. TheCMV-IE promoter is advantageously of murine or human origin. Thepromoter of the elongation factor 1α can also be used. A muscle specificpromoter can also be used (Li et al., 1999). Strong promoters are alsodiscussed herein in relation to plasmid vectors. In one embodiment, asplicing sequence can be located downstream of the enhancer/promoterregion. For example, the intron 1 isolated from the CMV-IE gene(Stenberg et al., 1984), the intron isolated from the rabbit or humanβ-globin gene, especially the intron 2 from the b-globin gene, theintron isolated from the immunoglobulin gene, a splicing sequence fromthe SV40 early gene or the chimeric intron sequence isolated from thepCI vector from Promege Corp. comprising the human β-globin gene donorsequence can be fused to the mouse immunoglobulin acceptor sequence(from about nucleotide 890 to about nucleotide 1022 in Genbank under theaccession number CVU47120). A poly(A) sequence and terminator sequencecan be inserted downstream the polynucleotide to be expressed, e.g. abovine growth hormone gene, especially from about nucleotide 2339 toabout nucleotide 2550 in Genbank under the accession number BOVGHRH, arabbit β-globin gene or a SV40 late gene polyadenylation signal.

In another embodiment the viral vector is a canine adenovirus,especially a CAV-2 (see, e.g. Fischer et al., 2002; U.S. Pat. Nos.5,529,780 and 5,688,920; PCT Application No. WO95/14102). For CAV, theinsertion sites can be in the E3 region and/or in the region locatedbetween the E4 region and the right ITR region (see U.S. Pat. Nos.6,090,393 and 6,156,567). In one embodiment the insert is under thecontrol of a promoter, such as a cytomegalovirus immediate-early genepromoter (CMV-IE promoter) or a promoter already described for a humanadenovirus vector. A poly(A) sequence and terminator sequence can beinserted downstream the polynucleotide to be expressed, e.g. a bovinegrowth hormone gene or a rabbit β-globin gene polyadenylation signal.

In another embodiment, the viral vector is a herpesvirus such as acanine herpesvirus (CHV) or a feline herpesvirus (FHV). For CHV, theinsertion sites may be in the thymidine kinase gene, in the ORF3, or inthe UL43 ORF (see U.S. Pat. No. 6,159,477). In one embodiment thepolynucleotide to be expressed is inserted under the control of apromoter functional in eukaryotic cells, advantageously a CMV-IEpromoter (murine or human). A poly(A) sequence and terminator sequencecan be inserted downstream the polynucleotide to be expressed, e.g.bovine growth hormone or a rabbit β-globin gene polyadenylation signal.

For recombinant vectors based on a poxvirus vector, a vaccinia virus oran attenuated vaccinia virus, (for instance, MVA, a modified Ankarastrain obtained after more than 570 passages of the Ankara vaccinestrain on chicken embryo fibroblasts; see Stickl & Hochstein-Mintzel,Munch. Med. Wschr., 1971, 113, 1149-1153; Sutter et al., Proc. Natl.Acad. Sci. U.S.A., 1992, 89, 10847-10851; available as ATCC VR-1508; orNYVAC, see U.S. Pat. No. 5,494,807, for instance, Examples 1 to 6 and etseq of U.S. Pat. No. 5,494,807 which discuss the construction of NYVAC,as well as variations of NYVAC with additional ORFs deleted from theCopenhagen strain vaccinia virus genome, as well as the insertion ofheterologous coding nucleic acid molecules into sites of thisrecombinant, and also, the use of matched promoters; see alsoWO-A-96/40241), an avipox virus or an attenuated avipox virus (e.g.,canarypox, fowlpox, dovepox, pigeonpox, quailpox, ALVAC or TROVAC; see,e.g., U.S. Pat. Nos. 5,505,941, 5,494,807) can be used. Attenuatedcanarypox viruses are described in U.S. Pat. No. 5,756,103 (ALVAC) andWO-A-01/05934. Reference is also made to U.S. Pat. No. 5,766,599 whichpertains to the attenuated fowlpox strain TROVAC. Reference is made tothe canarypox available from the ATCC under access number VR-111.Numerous fowlpox virus vaccination strains are also available, e.g. theDIFTOSEC CT strain marketed by MERIAL and the NOBILIS VARIOLE vaccinemarketed by INTERVET. For information on the method used to generaterecombinants thereof and how to administer recombinants thereof, theskilled artisan can refer documents cited herein and to WO-A-90/12882,e.g., as to vaccinia virus mention is made of U.S. Pat. Nos. 4,769,330,4,722,848, 4,603,112, 5,110,587, 5,494,807, and 5,762,938 inter alia; asto fowlpox, mention is made of U.S. Pat. Nos. 5,174,993, 5,505,941 and5,766,599 inter alia; as to canarypox mention is made of U.S. Pat. No.5,756,103 inter alia. When the expression vector is a vaccinia virus,insertion site or sites for the polynucleotide or polynucleotides to beexpressed are advantageously at the thymidine kinase (TK) gene orinsertion site, the hemagglutinin (HA) gene or insertion site, theregion encoding the inclusion body of the A type (ATI); see alsodocuments cited herein, especially those pertaining to vaccinia virus.In the case of canarypox, advantageously the insertion site or sites areORF(s) C3, C5 and/or C6; see also documents cited herein, especiallythose pertaining to canarypox virus. In the case of fowlpox,advantageously the insertion site or sites are ORFs F7 and/or F8; seealso documents cited herein, especially those pertaining to fowlpoxvirus. The insertion site or sites for MVA virus area advantageously asin various publications, including Carroll M. W. et al., Vaccine, 1997,15 (4), 387-394; Stittelaar K. J. et al., J. Virol., 2000, 74 (9),4236-4243; Sutter G. et al., 1994, Vaccine, 12 (11), 1032-1040; and, inthis regard it is also noted that the complete MVA genome is describedin Antoine G., Virology, 1998, 244, 365-396, which enables the skilledartisan to use other insertion sites or other promoters. Advantageously,the polynucleotide to be expressed is inserted under the control of aspecific poxvirus promoter, e.g., the vaccinia promoter 7.5 kDa (Cochranet al., J. Virology, 1985, 54, 30-35), the vaccinia promoter 13L(Riviere et al., J. Virology, 1992, 66, 3424-3434), the vacciniapromoter HA (Shida, Virology, 1986, 150, 451-457), the cowpox promoterATI (Funahashi et al., J. Gen. Virol., 1988, 69, 35-47), the vacciniapromoter H6 (Taylor J. et al., Vaccine, 1988, 6, 504-508; Guo P. et al.J. Virol., 1989, 63, 4189-4198; Perkus M. et al., J. Virol., 1989, 63,3829-3836), inter alia.

In a further embodiment, the recombinant viral vector is the recombinantALVAC canarypox virus vCP2350, as described in the example 3.

The vaccines containing recombinant viral vectors according to theinvention may be freeze-dried, advantageously with a stabiliser.Freeze-drying can be done according to well-known standard freeze-dryingprocedures. The pharmaceutically or veterinary acceptable stabilisersmay be carbohydrates (e.g. sorbitol, mannitol, lactose, sucrose,glucose, dextran, trehalose), sodium glutamate (Tsvetkov T et al.,Cryobiology 1983, 20(3): 318-23; Israeli E et al., Cryobiology 1993,30(5): 519-23), proteins such as peptone, albumin, lactalbumin orcasein, protein containing agents such as skimmed milk (Mills C K etal., Cryobiology 1988, 25(2): 148-52; Wolff E et al., Cryobiology 1990,27(5): 569-75), and buffers (e.g. phosphate buffer, alkaline metalphosphate buffer). An adjuvant may be used to make soluble thefreeze-dried preparations.

Any vaccine composition according to the invention can alsoadvantageously contain one or more adjuvanta.

For the Plasmids:

The plasmid-based vaccines can be formulated with cationic lipids,adventageously with DMRIE(N-(2-hydroxyéthyl)-N,N-diméthyl-2,3-bis(tetradecyloxy)-1-propanammonium;WO-A-96/34109), and adventageously in association with a neutral lipid,for example DOPE (dioleoyl-phosphatidyl-ethanolamine; Behr J. P.,Bioconjugate Chemistry, 1994:5:382-389), in order to form DMRIE-DOPE. Inone embodiment, the mixture is made extemporaneously, and before itsadministration it is advantageous to wait about 10 min to about 60 min,for example, about 30 min, for the appropriate complexation of themixture. When DOPE is used, the molar ratio of DMRIE/DOPE can be from95/5 to 5/95 and is advantageously 1/1. The weight ratio plasmid/DMRIEor DMRIE-DOPE adjuvant is, for example, from 50/1 to 1/10, from 10/1 to1/5 or from 1/1 to 1/2.

Optionally a cytokine can be added to the composition, especially GM-CSFor cytokines inducing Th1 (e.g. IL12). These cytokines can be added tothe composition as a plasmid encoding the cytokine protein. In oneembodiment, the cytokines are from canine origin, e.g. canine GM-CSFwhich gene sequence has been deposited at the GenBank database(accession number S49738). This sequence can be used to create saidplasmid in a manner similar to what was made in WO-A-00/77210(incorporated herein by reference).

For the Recombinant Viral Vectors:

The recombinant viral vector-based vaccine can be combined withfMLP(N-formyl-methionyl-leucyl-phenylalanine; U.S. Pat. No. 6,017,537incorporated herein by reference) and/or Carbomer adjuvant (PhameuropaVol. 8, No. 2, June 1996). Persons skilled in the art can also refer toU.S. Pat. No. 2,909,462 (incorporated herein by reference) whichdescribes such acrylic polymers cross-linked with a polyhydroxylatedcompound having at least 3 hydroxyl groups, advantageously not more than8, the hydrogen atoms of at least three hydroxyls being replaced byunsaturated aliphatic radicals having at least 2 carbon atoms. Forexample, the radicals are those containing from 2 to 4 carbon atoms,e.g. vinyls, allyls and other ethylenically unsaturated groups. Theunsaturated radicals may themselves contain other substituents, such asmethyl. The products sold under the name Carbopol® (BF Goodrich, Ohio,USA) are appropriate. The products are cross-linked with an allylsucrose or with allyl pentaerythritol. Among them, there may beadvantageously mentioned Carbopol® 974P, 934P and 971P.

Among the copolymers of maleic anhydride and alkenyl derivative, thecopolymers EMA® (Monsanto) which are copolymers of maleic anhydride andethylene, linear or cross-linked, for example cross-linked with divinylether, are advantageous. Reference may be made to J. Fields et al.,Nature, 186: 778-780, 4 Jun. 1960, incorporated herein by reference.

The polymers of acrylic or methacrylic acid and the copolymers EMA® areformed, for example, of basic units of the following formula:

in which:

-   -   R₁ and R₂, which are identical or different, represent H or CH₃    -   x=0 or 1, preferably x=1    -   y=1 or 2, with x+y=2

For the copolymers EMA®, x=0 and y=2. For the carbomers, x=y=1.

The dissolution of these polymers in water leads to an acid solution,which is neutralized, adventageously to physiological pH, in order toprovide the adjuvant solution into which the vaccine itself isincorporated. The carboxyl groups of the polymer are then partly in COO⁻form.

In one embodiment, a solution of adjuvant, especially of carbomer, isprepared in distilled water, advantageously in the presence of sodiumchloride, the solution obtained being at an acidic pH. This stocksolution is diluted by adding it to the desired quantity (for obtainingthe desired final concentration), or a substantial part thereof, ofwater charged with NaCl, adventageously physiological saline (NaCl 9g/l) all at once in several portions with concomitant or subsequentneutralization (pH 7.3 to 7.4), adventageously with NaOH. This solutionat physiological pH is used for mixing with the vaccine, which may beespecially stored in freeze-dried, liquid or frozen form.

The polymer concentration in the final vaccine composition can be from0.01% to 2% w/v, from 0.06 to 1% w/v, or from 0.1 to 0.6% w/v.

Another aspect of the present invention is methods of prime-boostvaccination of Leishmania-susceptible subjects using the vaccinecompositions according to the invention.

By definition, Leishmania-susceptible subjects encompass humans, felids(i.e. domesticated cats, kittens, big cats and wild cats, for example)and canids (i.e. dogs, bitchs, puppies, foxes, jackals, and wolves, forexample). In one embodiment, canines are a suitable subject foradministration of the vaccine according to the present invention.

These prime-boost administration methods include at least two differentadministrations, which consist of at least one primo-administration ofan effective amount of a vaccine composition according to the inventionand after a certain period of time at least one boost administration ofan effective amount of a vaccine composition according to the invention,wherein 1) the vaccine compositions for the primo-administration areplasmid-based vaccines and the vaccine compositions for the boostadministration are recombinant viral vector-based vaccines; and/or 2)the vaccine compositions for the primo-administration are plasmid-basedvaccines coupled to electrotransfer treatment and the vaccinecompositions for the boost administration are recombinant viralvector-based vaccines; and/or 3) the vaccine compositions are the samefor the primo-administration and for the boost administration and theroute of administration and/or the means of administration are not thesame for the primo-administration and for the boost administration. Theroutes of administration can be, for example, intramuscular (IM) orintradermal (ID) or subcutaneous. The means of administration can be,for example, a syringe with a needle, or needle free apparatus, or asyringe with a needle coupled to electrotransfer (ET) treatment, orneedle free apparatus coupled to ET treatment.

The prime-boost administrations are advantageously carried out 2 to 6weeks apart, for example, about 3 weeks apart. According to oneembodiment, a semi-annual booster or an annual booster, advantageouslyusing the viral vector-based vaccine, is also envisaged. The animals areadvantageously at least 6 to 8 weeks old at the time of the firstadministration.

Another embodiment of the prime-boost administration regimen consists ofprimo-administration of a plasmid-based vaccine and a boostadministration of a recombinant poxvirus vector-based vaccine, forexample, with a canarypox virus vector. Both priming and boostingadministrations are advantageously done via intradermal (ID) route usinga needle free apparatus. In one embodiment, this plasmid-based vaccineis a vaccine comprising pVR1020 KMP11 as described in example 1, andthis canarypox virus vector is vCP2350 as described in example 3.

In yet another embodiment of the present invention, theprimo-administration is made with a plasmid-based vaccine via the IDroute using a needle free apparatus and the boost administration is madewith a plasmid-based vaccine via the intramuscular (IM) route using asyringe and a needle coupled to ET treatment. In a further embodiment,this plasmid-based vaccine is a vaccine comprising pVR1020KMP11 asdescribed in example 1.

A further embodiment of the prime-boost administration regimen consistsof primo-administration of a plasmid-based vaccine coupled to ETtreatment and boost administration of a recombinant poxvirusvector-based vaccine, advantageously with a canarypox virus vector. Theprimo-administration is advantageously done intramuscularly and theboost administration is advantageously done via intradermal (ID) routeusing a needle free apparatus. In another embodiment, this plasmid-basedvaccine is a vaccine comprising pVR1020KMP11 as described in example 1,and this canarypox virus vector is vCP2350 as described in example 3.

In another embodiment, the primo-administration comprises aplasmid-based vaccine via intramuscular (IM) route using a syringe and aneedle coupled to ET treatment and the boost administration comprises aplasmid-based vaccine via ID route using a needle free apparatus. In oneembodiment, this plasmid-based vaccine is a vaccine comprisingpVR1020KMP11 as described in example 1.

In one embodiment of the prime-boost regimen, the primo-administrationutilizes a needle free apparatus via the ID route with a plasmid-basedvaccine, a first boost administration comprises a plasmid-based vaccinevia IM route using a syringe and a needle coupled to ET treatment, and asecond boost administration utilizes a needle free apparatus via IDroute with a recombinant viral vector-based vaccine, advantageously witha canarypox vector. In a further embodiment, this plasmid-based vaccineis a vaccine comprising pVR1020KMP11 as described in example 1, and thiscanarypox virus vector is vCP2350 as described in example 3.

Another aspect of the invention is the use of a plasmid-based vaccineaccording to the present invention for administration toLeishmania-susceptible animals, wherein this administration is coupledto ET treatment. The administration of a plasmid-based vaccine isadvantageously intramuscular. The means of administration is, forexample, a syringe and a needle. One or several injections can beadministered successively. In the case of several injections, they canbe carried out 2 to 6 weeks apart, for example, about 3 weeks apart.According to one embodiment, a semi-annual booster or an annual boosteris also envisaged.

For plasmid-based vaccines, an advantageousroute of administration isID. This administration can be made by a syringe with a needle or with aneedle free apparatus like Dermojet or Biojector (Bioject, Oreg., USA)or Vetjet™ (Merial) or Vitajet™ (Bioject Inc.), see US-A-2006/0034867.The dosage can be from 50 μg to 500 μg per plasmid. When DMRIE-DOPE isadded, 100 μg per plasmid can be utilized. When canine GM-CSF or othercytokines are used, the plasmid encoding this protein is present at adosage of from about 200 μg to about 500 μg and can advantageously be200 μg. The volume of doses can be between 0.01 ml and 0.5 ml, forexample, 0.25 ml. Administration can be provided with multiple points ofinjection.

Another envisioned route of administration for plasmid-based vaccines isthe IM route coupled to electrotransfer (ET) treatment. The ET treatmentcan be performed using an apparatus for electrotransfer and thespecifications of the manufacturer (i.e. Sphergen G250 generator(Sphergen SARL, Evry Genopole, France); MedPulser® DNA electroporationsystem (Innovio Biomedical Corporation, San Diego, Calif., USA)). In oneembodiment, the apparatus for electrotransfer has a unipolar field. Thefield intensity can be from about 50 to about 250 V/cm, from about 50 toabout 200 V/cm, or from about 50 to about 175 V/cm. The pulse durationis from about 1 to about 50 msec, or from about 15 to about 25 msec. Thefrequency is from about 1 to about 50 Hz, or from about 5 to about 15Hz. The interpulse interval is from about 1 to 1000 msec, or from about1 to about 200 msec. The number of pulses is from 1 to 20, or from 5 to10. The intra tissular intensity is advantageously up to about 2 A. Thedistance between electrodes is from about 0.2 to about 1 cm, or fromabout 0.2 to about 0.5 cm.

For recombinant viral vector-based vaccines, the route of administrationis advantageously ID. This administration can be made by a syringe witha needle or with a needle free apparatus like Dermojet or Biojector(Bioject, Oreg., USA) or Vetjet™ (Merial) or Vitajet™ (Bioject Inc.).The dosage is from about 10³ pfu to about 10⁹ pfu per recombinantpoxvirus vector. When the vector is a canarypox virus, the dosage is,for example, from about 10⁵ pfu to about 10⁹ pfu, or from about 10⁶ pfuto about 10⁸ pfu. The volume of doses is from about 0.01 ml to 0.2 ml,and is advantageously 0.1 ml. Administration can comprise multiplepoints of injection.

For the IM route the volume of the vaccine provided is from 0.2 to 2 ml,or from about 0.5 to 1 ml. The same dosages are utilized for any of thevectors of the present invention.

Another aspect of the present invention is a kit for prime-boostvaccination according to the present invention. The kit comprises atleast two vials: a first vial containing a vaccine for theprimo-vaccination according to the present invention, and a second vialcontaining a vaccine for the boost-vaccination according to the presentinvention. The kit can advantageously contain additional first or secondvials for additional primo-vaccinations or additionalboost-vaccinations.

In one embodiment, the kit comprises two vials, one containing aplasmid-based vaccine for the primo-vaccination according to the presentinvention, the other vial containing a recombinant viral vector-basedvaccine for the boost-vaccination according to the present invention.

In another embodiment, the kit comprises two vials, one containing apVR1020KMP11 plasmid-based vaccine for the primo-vaccination accordingto the present invention, the other vial containing a vCP2350vector-based vaccine for the boost-vaccination according to the presentinvention.

The invention will now be further described by way of the followingnon-limiting examples.

EXAMPLES

Without further elaboration, it is believed that one skilled in the artcan, using the preceding descriptions, practice the present invention toits fullest extent. The following detailed examples are to be construedas merely illustrative, and not limitations of the preceding disclosurein any way whatsoever. Those skilled in the art will promptly recognizeappropriate variations from the procedures both as to reactants and asto reaction conditions and techniques.

Construction of DNA inserts, plasmids, recombinant viral vectors wascarried out using the standard molecular biology techniques described byJ. Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). All therestriction fragments used for the present invention were isolated usingthe “Geneclean” kit (BIO 101 Inc., La Jolla, Calif.).

Example 1 Construction of a Plasmid Expressing the L. Infantum KMP11Antigen

The nucleic acid sequence encoding the L. infantum KMP11 was synthesizedchemically, having the sequence described in SEQ ID NO: 1 and havingpolyadenine tails. The KMP11 fragment was amplified by PCR and clonedinto the TOPO cloning site of the pVR2001-TOPA (or pVR2001-TOPO)(Oliveira F. et al. Vaccine (2006) 24: 374-90), having the tissueplasminogen activator signal peptide (TPA), to obtain the plasmidpVR1020KMP11. VR2001-TOPO is derived from the plasmid VR1020. VR1020 isa plasmid backbone available from Vical, Inc., (Sandiego, Calif.) whichhas been previously used, see, e.g., U.S. Pat. Nos. 6,451,769 and7,078,507; as described in Oliveira et al., plasmid VR2001-TOPO (orpVR2001-TOPA) is VR1020 modified by the addition of topoisomerasesflanking the cloning site and containing coding for and expressing asignal secretory peptide that increases the likelihood of producing asecreted protein (see FIG. 1 in Oliveira F. et al. Vaccine (2006) 24:374-90).

The nucleic acid sequence of one strand of the plasmid pVR1020KMP11 isdescribed in SEQ ID NO: 5, and the plasmid map is provided in FIG. 10.In accordance with the present invention, VR1020KMP11 therefore containsand expresses a DNA encoding a promoter for driving expression in amammalian cell, a DNA encoding a leader peptide for facilitatingsecretion/release of a prokaryotic protein sequence from a mammaliancell, for example a CMV promoter, a DNA encoding KMP11, and a DNAencoding a terminator. VR1020KMP11 additionally contains topoisomerasesflanking the DNA encoding KMP11 and containing coding for a signalsecretory peptide that increases the likelihood of producing a secretedprotein.

Example 2 Prime-Boost Vaccination of Dogs Against Leishmaniasis withPlasmid-Based Vaccines

Two groups of 5 conventional dogs were vaccinated either with a plasmidexpressing the L. infantum KMP11 antigen or with the control plasmidhaving no insert. Priming administration was performed intradermally atD0 using the Merial Vetjet™ needle free injector.

The Merial Vetjet™ uses compressed air as a power source. Theneedle-free injection leads to reduced lesions and trauma at theinjection site when compared to current needle usage. The device isactivated by placing the nozzle against the subject and will not fireuntil the mechanism travels approximately 0.30″ against a spring load.

The Merial Vetjet™ has three basic steps in the operation cycle: (1)pull the trigger, (2) push the device against the target injection siteand (3) pull the device away from the injection site and release thetrigger. When the trigger is pulled, the trigger activates the initiatorvalve. The trigger sets the firing mechanism into the priming position.When the device is pushed against the target injection site, the firingmechanism slides back and releases the poppet valve and the drug isexpelled through the nozzle. When the device is pulled away from theinjection site and the trigger is released, the firing mechanismretracts. Exhaust air is expelled, activating the dye system (ifdesired). The drug fill system loads a fresh dose.

The inner thigh area of the two groups of 5 conventional dogs wasclipped free of fur and disinfected with povidone iodine (Vetedine®,Vetoquinol, Lure, France) before administration. Vaccinated dogs (n=5)received in the right medial thigh a dose of 400 μg of purified pVR1020KMP11 plasmid (see Example 1) in a volume of 0.2 ml of TE pH 8 buffer(i.e., 2 mg/ml). Control dogs (n=5) received in a similar manner theempty expression plasmid pVR1020. Dogs were not anesthetized for thisadministration.

A booster administration was performed at D21 using the same plasmids asfor the priming (i.e., pVR1020KMP11 in vaccinated dogs and pVR1020 incontrol dogs) and the same dose (400 μg at 2 mg/ml), but using anintramuscular delivery coupled to electrotransfer (ET) treatment. Dogswere anesthetized by the intramuscular route using 5 mg/kg ketamine and10 mg/kg medetomidine.

The IM administration and ET treatment was applied to the left innerthigh area (i.e., left semi-membranous muscle). The area was clippedfree of fur and disinfected with povidone iodine (Vetedine®) beforetreatment. The injection was performed using the Sphergen 3 needledevice INJ-1 (Sphergen SARL, Evry Genopole, France) with needlesseparated by 0.5 cm each. The ET treatment will take place immediatelyfollowing the injection, using a Sphergen G250 generator (Sphergen SARL,Evry Genopole, France). The following ET specifications were used: T1:20 msec, T2: 80 msec, frequency: 10 Hz, 10 pulses. Applied voltage was87.5 V, creating an electric field of 175 V/cm within the muscle.

At D0, D14, D41, D55 and D76, sera were analyzed for frequency of CD8′cells (see FIG. 1), frequency of interferon-γ-producing (IFN-γ) cells(see FIG. 2), specific CD8 T cell proliferation (see FIG. 3) andspecific CD4 T cell proliferation (see FIG. 4).

IFN-γ Production by Cultured Whole Blood

The whole blood was diluted 1:8 in RPMI supplemented with 3%antibiotic/antimycotic solution (GIBCO, Grand Island, N.Y., USA) in a48-well flat-bottomed culture plate with a final volume of 1 ml perwell. Cells were stimulated by the addition of 25 μg/ml of soluble L.infantum extract (SLE) of Phorbol 12-myristate 13-acetate (PMA,Sigma-Aldrich Co., USA). After 48 h of incubation at 37° C., 700 μl ofsupernatant was removed from each well and stored at −20° C. untilrequired for the cytokine assay. IFN-γ was measured using a captureELISA assay for dogs (R&D Systems, Minneapolis, Minn., USA) followingthe manufacturer's instructions. Biotin-labeled detection antibodieswere used, revealed with streptavidin-HRP (Amersham Biosciences) and TMBsubstrate (KPL, Gaithesburgh, Md., USA). The reaction was stopped by theaddition of Stop solution (KPL) to each well. Absorbance values are readat 450 nm in an automatic micro-ELISA reader (Thermo Multiskan EX,Waltham, Mass., USA).

Cellular Profile of Peripheral Blood and Cytokine Staining

The phenotyping of peripheral blood cell populations was performed usingwhole blood samples collected at the indicated days. In short, 1 ml ofblood was fixed and the erythrocytes lysed with fixative solution (10.0g/l paraformaldehyde; 10.2 g/l cacodylic acid; 6.65 g/l sodium chloride;pH 7.2). After 10-min incubation, cells were washed twice with PBS—1%BSA buffer. Surface staining was carried out in a 96-well round bottomedmicroplate with anti-canine CD4-Alexa 647 and anti-canine CD8-PE. Thedata on fluorescently labeled cells were acquired in a FACScalibur flowcytometer (Becton Dickinson, San Jose, Calif., USA). At least thirtythousand events were counted. Intracellular cytokine staining wascarried out after permeabilization with saponin for the IFN-γ usinganti-bovine IFN-γ-PE (Serotec).

Lymphoproliferation of Peripheral Blood Leukocytes (PBLs)

The lymphoproliferation assay was performed using PBLs obtained afterseparation with ficoll. PBMCs were diluted in 1 ml phosphate-bufferedsaline (PBS) and stained with the fluorescent dye carboxyfluoresceindiacetate succinimidyl ester (CFSE, Molecular probes, Carlsbad, Calif.,USA). After staining, cells were resuspended in 200 μl RPMI supplementedwith 10% FBS and 3% antibiotic/antimycotic solution (GIBCO, GrandIsland, N.Y., USA). All tests were performed in triplicate in 96-wellflat-bottomed culture plates using SLE at a concentration of 25 μg/mland Concanavalin A (ConA) (Sigma-Aldrich Co., USA) at 160 μg/ml.Incubation was carried out in a humidified 5% CO₂ atmosphere at 37° C.for 5 days. Cells were then collected and stained for CD4 or CD8expression as described above. Percentage of proliferation was assessedby loss of fluorescent intensity in these populations.

Serological Analysis

Blood was collected at different time points, and the serum wasseparated and stored frozen at −20° C. Antigen-specific canine IgG, IgG₁and IgG₂ were measured by indirect enzyme-linked immunosorbent assay(ELISA). Briefly, the antigens were coated onto 96-well microplates(MaxiSorp™, Nalge Nunc, Rochester, N.Y., USA) at a concentration of 8μg/ml for SLE and at 5 μg/ml for the recombinant antigens. Sera wereadded at the concentrations 1:100, 1:500 and three-fold serial dilutionsthereafter followed by washes and addition of peroxidase-conjugatedanti-dog IgG, IgG_(i) and IgG₂ (Bethyl Laboratories Inc., Montgomery,Tex., USA) at 1:8000, 1:1500 and 1:3000 dilution, respectively. Wellswere then washed and substrate and chromogen (TMB, KPL) were added andabsorbance was read on an automatic ELISA microplate reader at 450 nm.The mean optical density of control canine sera was used as a baseline.The last serum dilution greater than three times above baseline wasconsidered the titration endpoint. The geometric mean of these endpointswas calculated for the five dogs from each group.

Western Blot Analysis

Western blot analysis was performed using SLE (0.6 mg/ml), which wereboiled in SDS sample buffer, separated on a 4-12% gradient Tris-GlycineSDS-PAGE gel (Invitrogen, Cal) and transferred onto a PVDF membrane(Pall Life Sciences, East Hills, N.Y.). The blot was blocked overnightwith blocker solution (Invitrogen, Carlsbad, Calif., USA) and probedwith each individual serum diluted 1:1000. Horseradishperoxidase-conjugated anti-dog was used as a secondary antibody at adilution 1:2000. Development was performed with ECL reagent (AmershamBiosciences, Buckinghamshire, England) according to the manufacturer'sinstructions.

FIG. 1 shows a significant difference in the total of CD8 T cellspresent in the peripheral blood after the boost administration betweenthe sera of the KMP11 vaccinated dogs and those of the control dogs(p=0.01).

The FIG. 2 shows a significant difference in the total ofIFN-γ-producing cells present in the peripheral blood after theprimo-administration and after the boost administration between the seraof the KMP11 vaccinated dogs and those of the control dogs (p=0.04).

FIG. 3 shows a significant difference in the total of specificanti-KMP11 CD8 T cells present in the peripheral blood before the boostadministration (3 weeks after priming) and after the boostadministration between the sera of the KMP11 vaccinated dogs and thoseof the control dogs (p=0.007).

FIG. 4 shows a significant difference in the total of specificanti-KMP11 CD4 T cells present in the peripheral blood before the boostadministration (3 weeks after priming) and after the boostadministration between the sera of the KMP11 vaccinated dogs and thoseof the control dogs (p=0.03).

At D0, D14, D41, D55 and D76, sera were also analyzed for antibodyresponses by Western Blot using L. infantum lysate at 6 μg/10 μA perlane (see FIG. 5). Sera were tested at a dilution of 1:1000. Thesecondary antibody was HRP anti-canine IgG reagent used at a dilution of1:2000. The Western Blot results depicted in FIG. 5 show a clearpost-boost KMP11-specific IgG response (see circled bands).

Example 3 Construction of an ALVAC Canarypox Virus Vector Expressing theL. infantum KMP11 Antigen

The nucleotide insert used in the construction of vCP2350 was derivedfrom the L. infantum KMP11 gene supplied by GeneArt GmbH (Regensburg,Germany). The nucleic acid sequence is synthetic with codon optimizationfor expression in mammalian cells (SEQ ID NO: 3). This nucleic acidsequence encodes L. infantum KMP11 antigen (SEQ ID NO: 4). Two differentenzyme restriction sites flanked this nucleotide insert, an EcoRV site5′ of the coding region and a XbaI site 3′ of the coding region.

To construct the ALVAC donor plasmid, pALVAC C₅H₆p-leishmania-11(pJSY1992.1), the nucleotide insert was digested using EcoRV/XbaIdigestion in order to isolate the fragment comprising the syntheticKMP11 gene. (For discussion and examples of the plasmid, pALVAC, and theC5 locus, see e.g., U.S. Pat. Nos. 5,756,103; 5,833,975; and 6,780,407).The sequence of the vaccinia virus H6 promoter has been previouslydescribed (see e.g., Taylor et al. Vaccine. 6: 497-503, 1988a; Taylor etal. Vaccine. 6: 504-508, 1988b; Guo et al. J Virol 63: 4189-4198, 1989).

This fragment was then ligated to EcoRV/XbaI digested pALVAC C5 H6pdonor (pCXL148.2). The resulting plasmid pJSY1992.1 (FIG. 6) wassequenced (SEQ ID NO: 6) and confirmed to contain the correct nucleicacid sequence (SEQ ID NO: 3) of the KMP11 gene.

To generate vCP2350, plasmid pJSY1992.1, which contained the syntheticKMP11 gene, was linearized with NotI restriction enzyme. The linearizedfragments were individually transfected into ALVAC-infected primary CEFcells by using the calcium phosphate precipitation method describedpreviously (Panicali et al., Proc. Natl. Acad Sci USA, 1982, 79:4927-4931; Piccini et al., Methods Enzymol., 1987, 153: 545-563). After24 h, the transfected cells were harvested, sonicated and used forrecombinant virus screening.

Recombinant plaques were screened based on the plaque lift hybridizationmethod using a Leishmania synthetic KMP11-specific probe which waslabeled with horse radish peroxidase according to the manufacturer'sprotocol (Amersham Cat# RPN-3001). After three sequential rounds ofplaque purification, the recombinants, designated as vCP2350.1.1.5, weregenerated and confirmed by hybridization as 100% positive for theLeishmania synthetic KMP11 insert and 100% negative for the C5 ORF.

A single plaque was selected from the third round of plaque purificationand expanded to obtain P1 (60 mm), P2 (T75 flasks), P3 (roller bottles)stocks to amplify vCP2350.1.1.5. The infected cell culture fluid fromthe roller bottles was harvested and concentrated to produce virus stock(about 3.5 mL at 1.3×10¹⁰ PFU/mL).

A theoretical restriction enzyme gel for the genomic DNA was created inVector NTI and is shown in FIG. 7. Genomic DNA was extracted fromvCP2350.1.1.5 and digested with BgIII, HindIII and PstI, and separatedby 0.8% agarose gel electrophoresis. The results showed the correctinsertion of the foreign gene sequence (see FIG. 8).

The genomic DNA digested with BgIII, HindIII and PstI was transferred tonylon membrane and Southern blot analysis was performed by probing withLeishmania synthetic KMP11 probe. Bands were observed at the expectedsizes, indicating the correct insertion of Leishmania synthetic KMP11into the C5 locus (FIG. 9).

A more detailed analysis of the P3 stock genomic DNA was performed byPCR amplification and sequence analysis of the flanking arms of the C5locus and the Leishmania synthetic KMP11 insert. Primers 7931.DC (SEQ IDNO: 7) and 7932.DC (SEQ ID NO: 8) located beyond the arms of the C5locus were used to amplify the entire C5R-Leishmania synthetic KMP11insert-C5L fragment. The results showed that the sequence of theLeishmania synthetic KMP11 insert and the C5 left and right arms aroundthe Leishmania synthetic KMP11 insert in vCP2350.1.1.5 were correct.

Recombinant vectors in accordance with the present invention cantherefore comprise a recombinant avipox virus, eg canarypox virusvector, such as a recombinant ALVAC vector, having a promoter fordriving expression, for example the H6 promoter, operably linked to DNAencoding KMP11, in a suitable site in the avipox virus genome such asthe C3 or C5 locus of canarypox (eg ALVAC).

Example 4 Prime-Boost Vaccination of Dogs Against Leishmaniasis withPlasmid-Based Vaccines and ALVAC Canarypox Virus Vector-Based Vaccines

At week 17 after the primo-administration, the animals of the vaccinatedgroup of Example 2 were administered the ALVAC canarypox virus vCP2350vector vaccine of Example 3 in a volume of 0.2 ml of TE pH 8 buffer(i.e., 2 mg/ml).

At the same time, the animals of the control group of Example 2 wereadministered a vaccine comprising a control ALVAC canarypox virus vectorexpressing a CDV antigen (vCP258, see Example 19 of U.S. Pat. No.5,756,102) in a volume of 0.2 ml of TE pH 8 buffer (i.e., 2 mg/ml).

The dose of vaccine, vCP2350 vector vaccine for vaccinated animals andvCP258 vector vaccine for control animals, was 10⁸ PFU per animal. Theadministration was performed for each animal intradermally in the rightmedial thigh using the Merial Vetjet™ needle free injector. The innerthigh area was clipped free of fur and disinfected with povidone iodine(Vetedine®, Vetoquinol, Lure, France) before administration.

Sera of animals are collected and analyzed for frequency of CD8′ cells,frequency of IFN-γ-producing cells, specific CD8 T cell proliferationand specific CD4 T cell proliferation as described in Example 2.

A challenge with L. infantum amastigotes is scheduled at week 22 afterthe primo-administration.

The invention will now be further described by the following numberedparagraphs:

1. Use of in vivo expression vectors in a prime-boost administrationregimen, comprising a primo-administration of a vaccine comprising, in apharmaceutically acceptable vehicle, diluent or excipient, an in vivoexpression vector containing a polynucleotide sequence for expressing,in vivo, Leishmania KMP11 polypeptide, antigen, epitope or immunogen,followed by a boost administration of a vaccine comprising, in apharmaceutically acceptable vehicle or excipient, an in vivo expressionvector containing a polynucleotide sequence for expressing, in vivo,Leishmania KMP11 antigen, epitope or immunogen to protect canine animalsfrom leishmaniasis and/or to prevent disease progression in infectedcanine animals.

2. The use according to the paragraph 1, wherein the protection is shownby the prevention of the diffusion and implantation of the parasite intointernal organs of said canine animals.

3. The use according to the paragraph 1 for the production of a vaccinefor the induction of humoral immune response in canine animals againstKMP11.

4. The use according to the paragraph 3, wherein the induction in canineanimals is induction of anti-KMP11 IgG1.

5. The use according to any one of the paragraphs 1 to 4 for theproduction of a vaccine for the induction of cell-mediated immuneresponse in canine animals against KMP11.

6. The use according to the paragraph 5, wherein the induction in canineanimals is induction of CD8 T cell-mediated immune response againstKMP11.

7. The use according to the paragraph 5 or 6, wherein the induction incanine animals is induction of CD4 T cell-mediated immune responseagainst KMP11.

8. The use according to any one of the paragraphs 1 to 7, wherein the invivo expression vector for the primo-administration is a plasmid, andthe in vivo expression vector for the boost administration is arecombinant viral vector.

9. The use according to the paragraph 8, wherein the in vivo expressionvector for the primo-administration is a plasmid, and the in vivoexpression vector for the boost administration is a recombinantcanarypox virus vector.

10. The use according to the paragraph 9, wherein the in vivo expressionvector for the primo-administration is the plasmid pVR1020KMP11, and thein vivo expression vector for the boost administration is a recombinantcanarypox virus vector vCP2350.

11. The use according to any one of the paragraphs 1 to 10, wherein thepolynucleotide sequence encoding the Leishmania KMP11 is thecodon-optimized polynucleotide sequence SEQ ID No 3, encoding aLeishmania infantum KMP11 antigen SEQ ID No 4.

12. The use according to any one of the paragraphs 1 to 11, wherein theprimo-administration is coupled to electrotransfer treatment.

13. The use according to any one of the paragraphs 1 to 7, wherein thein vivo expression vectors for primo-administration and for boostadministration are plasmids and comprise a polynucleotide sequenceencoding a Leishmania KMP11 antigen or immunogen or an epitope thereof,and the primo-administration is done intradermally with a needle freeapparatus and the boost administration is done intramuscularly with asyringe and a needle and coupled to electrotransfer treatment.

14. The use according to any one of the paragraphs 1 to 7, wherein thein vivo expression vectors for primo-administration and for boostadministration are plasmids and comprise a polynucleotide sequenceencoding a Leishmania KMP11 antigen or immunogen or an epitope thereof,and the primo-administration is done intramuscularly with a syringe anda needle and coupled to electrotransfer treatment and the boostadministration is done intradermally with a needle free apparatus.

15. The use according to the paragraph 13 or 14, wherein the in vivoexpression vectors are the plasmid pVR1020KMP11.

16. The use according to the paragraph 13 or 14, wherein thepolynucleotide sequence encoding the Leishmania KMP11 is thecodon-optimized polynucleotide sequence SEQ ID No 3, encoding aLeishmania infantum KMP11 antigen SEQ ID No 4.

17. A kit for prime-boost administration regimen according to any one ofthe paragraphs 1 to 12, comprising at least two vials, one vialcontaining a vaccine for the primo-vaccination, the other(s) vial(s)containing a vaccine for the boost-vaccination.

18. The kit according to the paragraph 17, wherein the kit comprises twovials, one containing a plasmid-based vaccine for the primo-vaccination,the other vial containing a recombinant viral vector-based vaccine forthe boost-vaccination.

1-20. (canceled)
 21. A method of vaccinating a subject susceptible toLeishmania comprising a prime-boost administration regimen wherein saidregimen comprises a primo-administration of a vaccine comprising anexpression vector comprising a polynucleotide encoding a LeishmaniaKMP11 polypeptide, antigen, epitope or immunogen, followed by a boostadministration of a vaccine comprising an expression vector comprising apolynucleotide encoding a Leishmania KMP11 antigen, epitope orimmunogen, to protect the subject from leishmaniasis and/or to preventdisease progression in infected subject.
 22. The method of claim 21,wherein the vaccination prevents the diffusion and implantation of theparasite into internal organs of the subject.
 23. The method of claim 21wherein a humoral immune response against KMP11 is induced and/or acell-mediated immune response against KMP11 is induced.
 24. The methodof claim 23, wherein the production of anti-KMP11 IgG1 is induced. 25.The method of claim 23, wherein the response induced is a CD8′ Tcell-mediated immune response against KMP11 and/or a CD4′ Tcell-mediated immune response against KMP11.
 26. The method of claim 21,wherein the expression vector for the primo-administration is a plasmid,and wherein the expression vector for the boost administration is arecombinant viral vector.
 27. The method of claim 26, wherein therecombinant viral vector is a recombinant canarypox virus vector. 28.The method of claim 26, wherein the expression vector for theprimo-administration is plasmid pVR1020KMP11 or a plasmid having all ofthe identifying characteristics of pVR1020KMP11, and the expressionvector for the boost administration is a recombinant canarypox virusvector vCP2350 or a recombinant canarypox virus vector having all of theidentifying characteristics of vCP2350.
 29. The method of claim 21,wherein the polynucleotide encodes a Leishmania KMP11 polypeptide havingthe sequence as set forth in SEQ ID NO:4.
 30. The method of claim 21,wherein the polynucleotide has the sequence as set forth in SEQ ID NO:3.31. The method of claim 21, wherein the primo-administration is coupledto electrotransfer treatment.
 32. The method of claim 21, wherein theexpression vectors for primo-administration and for boost administrationare plasmids and comprise a polynucleotide encoding a Leishmania KMP11antigen or immunogen or an epitope thereof that elicits an immuneresponse in a dog against Leishmania, and wherein theprimo-administration is done intradermally with a needle free apparatusand the boost administration is done intramuscularly with a syringe anda needle and coupled to electrotransfer treatment.
 33. The method ofclaim 21, wherein the expression vectors for primo-administration andfor boost administration are plasmids and comprise a polynucleotidesequence encoding a Leishmania KMP11 antigen or immunogen or an epitopethereof that elicits an immune response in a dog against Leishmania, andwherein the primo-administration is done intramuscularly with a syringeand a needle and coupled to electrotransfer treatment and the boostadministration is done intradermally with a needle free apparatus. 34.The method of claim 32 or 33, wherein the expression vectors are theplasmid pVR1020KMP11 or a plasmid having all of the identifyingcharacteristics of pVR1020 KMP11.
 35. The method of claim 32 or 33,wherein the polynucleotide encodes a Leishmania KMP11 polypeptide havingthe sequence as set forth in SEQ ID NO:4.
 36. The method of claim 32 or33, wherein the polynucleotide has the sequence as set forth in SEQ IDNO:3.
 37. A kit for a prime-boost administration regimen of claim 21,comprising at least two vials, the first vial containing a vaccine forthe primo-vaccination, and the remaining vial(s) containing a vaccinefor the boost-vaccination.
 38. The kit of claim 37, wherein the kitcomprises two vials, one containing a plasmid-based vaccine for theprimo-vaccination, the other vial containing a recombinant viralvector-based vaccine for the boost-vaccination.